Establishment of high reciprocal connectivity between clonal cortical neurons is regulated by the Dnmt3b DNA methyltransferase and clustered protocadherins

Cell-lineage-dependent, high reciprocal connections between layer 4 excitatory neurons

We examined the synaptic connections between GFP-positive neuron pairs (P-P pairs), which were presumed to be clonal neuron pairs, and between nonclonal GFP-positive and -negative neuron pairs (P-N pairs) using dual whole-cell recordings from layer 4 at P9–11, when synaptogenesis between layer 4 excitatory neurons starts in the barrel cortex [35], and at P13–16 and P18–20. We selected neuron pairs with an intercellular distance 50 ?m within a barrel (Fig. 3a, b). Excitatory neurons in the layer 4 barrel consist of spiny stellate neurons and star pyramidal neurons. The recorded neurons visualized successfully by biocytin were mostly spiny stellate neurons (95%, n?=?75 neurons) with a few star pyramidal neurons (5%, n?=?4 neurons), indicating that the recorded neurons were excitatory neurons. In each age group, the GFP-positive neurons represented about 10% of all of the neurons in layer 4 within 200 ?m of the recorded neurons (Fig. 3c).

Synaptic connections between clonal and nonclonal neuron pairs in the barrel cortex. a Differential interference contrast image of a brain slice with two recording electrodes in a layer 4 barrel. Scale bar: 100 ?m. b Green fluorescent protein (GFP)-positive cells in layer 4. Dashed line indicates a barrel. Scale bar: 50 ?m. c Proportion of GFP-positive neurons in the analyzed area at each age. The number of tested columns is 8, 18, and 18 for P9–11, P13–16, and P18–20, respectively. P?=?0.51 (Kruskal–Wallis test). d Representative average traces (n?=?20) of action potentials evoked by brief depolarizing voltage pulses in one cell (pre) and resultant excitatory postsynaptic currents in the other (post) with one-way (upper) or reciprocal (lower) connections between GFP-positive excitatory neurons in layer 4 (P-P pair, left) and GFP-positive and -negative neurons (P-N pair, right) in P18–20 wild-type chimeric mice. e Percentage of P-P (left) and P-N (right) pairs with one-way (blue), reciprocal (magenta), or no (gray) connections; the number of recorded pairs is shown on each bar. Number of mice for P-P pairs: P9–11(n?=?7), P13–16 (n?=?10), P18–20 (n?=?12); P-N pairs: P9–11(n?=?6), P13–16 (n?=?11), P18–20 (n?=?9). *P??0.05, ***P??0.001: connection probabilities in different age groups (?² test with a 3?×?2 matrix after Bonferroni correction). f, g Developmental changes in the connection probability (f) and the proportion of reciprocal connections in connected pairs (reciprocity, g). **P??0.01: P-P versus P-N pairs at the same age (Fisher’s exact test)

We examined the connections between simultaneously recorded neurons in both directions. Figure 3d shows examples of one-way and reciprocal connections in P-P and P-N pairs. Among the P-P pairs, the proportion of three types of pairs (unconnected pairs and connected pairs with one-way connections, and reciprocal connections) changed significantly during the developmental stages examined, as tested using a ?² test with a 3?×?2 matrix after Bonferroni correction (P??0.05 for P9–11 versus P13–16; P??0.001 for P9–11 versus P18–20 and for P13–16 versus P18–20; Fig. 3e). At P9–11, about a third of the pairs showed one-way connections, while reciprocal connections were rare. The proportion of reciprocally connected pairs increased from P9–11 to P13–16, while the proportion of one-way-connected pairs remained almost unchanged. If the probability of connection in one direction is independent of the presence or absence of connection in the other direction, the probability of reciprocal connections is expected to be the square of the connection probability. The proportion of reciprocal connections at P13–16 (25%) was higher than the expected proportion (18%), suggesting that reciprocal connections were preferentially established from P9–11 to P13–16. Thereafter, the proportion of one-way-connected pairs decreased, whereas that of reciprocally connected pairs remained almost the same. Such clear, age-dependent changes were not observed among the P-N pairs (Fig. 3e). Although the proportion of pairs with one-way, reciprocal, and no connections among P-N pairs was almost the same as that among P-P pairs at P9–11 (?² test with a 3?×?2 matrix, P?=?0.89), it remained unchanged during the next periods (P?=?0.29 for P9–11 versus P13-16; P?=?0.92 for P13–16 versus P18–20).

To further characterize the developmental changes in these connections, we calculated the connection probability, defined as the number of detected connections divided by the total number of tested connections (Fig. 3f). The connection probability was not significantly different between P-P and P-N pairs at P9–11 (Fisher’s exact test, P?=?0.84). At P13–16, the probability in P-P pairs increased more than 2-fold (P?=?0.002) but that in P-N pairs remained almost the same; thus, P-P pairs connected more frequently than P-N pairs (P?=?0.004). Thereafter, the connection probability became indistinguishable between P-P and P-N pairs at P18–20 (P?=?0.68) as observed initially, owing to the reduction of one-way connections in P-P pairs (Fig. 3f). Thus, the connection probability appeared to increase transiently between clonal neuron pairs but remained almost unchanged between nonclonal neuron pairs during this period.

We also analyzed the reciprocity, which is the proportion of reciprocally connected pairs among connected pairs, and found clearly different developmental changes in the P-P versus P-N pairs. The reciprocity increased similarly in P-P and P-N pairs from P9–11 to P13–16. Thereafter, the reciprocity continued to increase in P-P pairs, whereas it remained almost unchanged in P-N pairs (Fig. 3?g). Consequently, at P18–20, reciprocal connections accounted for the majority of the connections between P-P pairs (83%) but for only 41% of those between P-N pairs (Fisher’s exact test, P?=?0.005; Fig. 3?g). Thus, most of the connections between clonal neurons became reciprocal by 3 weeks after birth. In P-P pairs, high connection probability at P13–16 and high reciprocity at P18–20 were commonly observed in most of the animals (Additional file 2: Figure S2).

We examined whether there was any change in the morphological or electrophysiological properties of the spiny stellate cells between P13–16 and P18–20 when the dynamic, developmental change of the connectivity was observed in P-P pairs. No significant changes were found in the dendritic morphology of layer 4 spiny stellate neurons from P13–16 to P18–20 except that the number of branches very close to the soma increased (Additional file 3: Figure S3A–D). The small increase of branches near the soma might contribute to the transient increase of synapses in P-P pairs. In either the GFP-positive or GFP-negative neurons in chimeric mice, the resting membrane potential and threshold of action potential generation did not change from P13–16 to P18–20 (Additional file 3: Figure S3E, F). The input resistance decreased during this developmental period, consistent with a previous study (Additional file 3: Figure S3G) [36].

To examine whether the different developmental changes in P-P and P-N pairs resulted from an impaired connection development in chimeric mice, we compared the synaptic connectivity in chimeric and nonchimeric mice. The discrimination of P-P pairs from P-N pairs was not possible in nonchimeric mice, because the clonal neurons were not GFP-labeled. However, most of the cell pairs sampled from nonchimeric mice were probably P-N pairs, because clonal neurons originating from a single neural stem cell accounted for only about 10% of the neurons. The proportion of pairs with one-way, reciprocal, and no connections in nonchimeric mice (Additional file 4: Figure S4) was similar to that observed among the P-N pairs in chimeric mice at both P13–16 (?² test with a 3?×?2 matrix, P?=?0.76) and P18–20 (P?=?0.17) (Fig. 3e), indicating that the neural connections developed normally in the chimeric mice.

The chimeric mice used were mostly generated by injecting iPS cells derived from C57BL/6 mice into BDF1 blastocysts, although we used chimeric mice originating from C57BL/6 blastocysts in some of the experiments conducted at P18–20. Thus, the GFP-positive and GFP-negative neurons were derived from different mouse strains in most experiments. Although this difference might have caused the connectivity to differ between P-P pairs and P-N pairs, the connectivity at P18–20 was similar in chimeric mice generated using C57BL/6 blastocysts versus BDF1 blastocysts (Additional file 5: Figure S5A, B). This result suggests that the differences in connectivity arose from differences in cell lineage rather than in the mouse strains used for the iPS cells and blastocysts.

The connectivity difference between P-P and P-N pairs might also be ascribed to the fact that iPS cells are occasionally missing some genes [37]. However, we found similar high proportions of reciprocal connections in the P-P pairs at P18–20 for three different iPS cell lines (Additional file 5: Figure S5C). These results strongly support the view that reciprocal connections are frequently formed between layer 4 excitatory neurons originating from the same stem cell during normal development.